Defining an embryonal rhabdomyosarcoma endotype

Abstract

Rhabdomyosarcoma (RMS) is the most common childhood soft-tissue sarcoma. The largest subtype of RMS is embryonal rhabdomyosarcoma (ERMS) and accounts for 53% of all RMS. ERMS typically occurs in the head and neck region, bladder, or reproductive organs and portends a promising prognosis when localized; however, when metastatic the 5-yr overall survival rate is ∼43%. The genomic landscape of ERMS demonstrates a range of putative driver mutations, and thus the recognition of the pathological mechanisms driving tumor maintenance should be critical for identifying effective targeted treatments at the level of the individual patients. Here, we report genomic, phenotypic, and bioinformatic analyses for a case of a 3-yr-old male who presented with bladder ERMS. Additionally, we use an unsupervised agglomerative clustering analysis of RNA and whole-exome sequencing data across ERMS and undifferentiated pleomorphic sarcoma (UPS) tumor samples to determine several major endotypes inferring potential targeted treatments for a spectrum of pediatric ERMS patient cases.

Keywords: embryonal rhabdomyosarcoma.

Figure 1.

MRI of the abdomen and pelvis. A multilobulated heterogeneous T₂ hyperintense and T₁ isointense intraluminal mass (red asterisk) was found arising from the posterior bladder base with the following sequences: axial T₁ radial VIBE with fat saturation +C on the pelvis (A) and sagittal radial VIBE with fat saturation (B). A T₁ hypointense and T₂ hyperintense lesion ventral to the anterior urethra was found with the following sequences: axial T₂ with fat saturation on the pelvis (C) and sagittal IR triggered (D).

Figure 2.

Histology sections. Hematoxylin and eosin (H&E) staining sections from a small biopsy revealing a small round blue cell tumor favor embryonal rhabdomyosarcoma (top) and a surgical resection revealing a botryoid pattern (bottom). Scale bars, (top) (left) 10 µM; (middle) 100 µM; (right) 100 µM; (bottom) (left) 10 µM; (right) 100 µM.

Figure 3.

Circos plot. Circos plots were generated using tumor DNA exome, normal DNA exome, and tumor RNA sequencing data. Exome data was analyzed for somatic point mutation, indel, and copy-number variation data, as denoted by the inner and middle rings, respectively. Gene expression from RNA sequencing data was plotted on the outer ring.

Figure 4.

Index case endotype unsupervised clustering of embryonal rhabdomyosarcoma (ERMS) and non-rhabdomyosarcoma soft-tissue sarcoma (NRSTS) samples with alveolar rhabdomyosarcoma (ARMS) samples harboring the PAX7:FOXO1 fusion gene as a control determined several endotypes. Clustering was performed using DNA and RNA sequencing from tumor samples from our IRB-approved CuReFAST initiative and cell lines (light blue), cell lines from Gene Expression Omnibus (GEO) (purple), PDX mouse models from Champions Oncology (light gray), cell lines from St. Jude Children's Research Hospital (blue), ARM patient's biopsies from National Cancer Institute (orange), ERMS patient's biopsies from NCI (dark green), PDX mouse models from the Jackson Laboratory (light green), canine samples from Flint Animal Cancer Center (pink), genetically engineered mouse models (red), and the patient's tumor sample (dark blue avatar). The legend below marks samples with known sex, age, primary tumor site, somatic mutations, and diagnosis. Gene expression (log₂ (TPM +1)) is shown below in a heatmap on a scale of 15 (red) to 0 (green). Samples without matched RNA-sequencing data were given a value 0 for all genes. Below the gene expression heatmap, samples with somatic mutations in our genes of interest are indicated by a black box. Unsupervised clustering was performed separately on the expression and somatic data within the legend (vertical dendrogram). Because we suspected a germline predisposition/syndrome for this patient, we kept germline mutations in the dendrogram but did not do the same for the other tumors as they were presumably somatic, noninherited cases.

Figure 5.

Drug validation for rhabdomyosarcoma cell lines. BYL-719, GSK1059615, CUDC-907, TC-KHNS11, BEZ-235, BKM-120, and entinostat were tested on RMS cell lines RD (blue), SMS-CTR (red), RMS559 (green), and U57810 (purple) at varying concentrations.